A host-centric morphological profiling approach to identify repurposed antiviral drugs.

Antiviral drug discovery has traditionally targeted viral proteins, while host-directed strategies remain underexplored. We present a systematic drug repurposing strategy that uses morphological profiling to identify host-targeting antivirals. Using cell painting, we demonstrate that SARS-CoV-2 infection can be accurately determined from the morphological profile of virus-infected cells. Moreover, morphological features reveal how host cells respond to viral exposure, offering insights into antiviral activity, host-cell health, and putative mechanisms of action of the compounds. Screening 5,275 repurposable compounds, we identified candidates that reversed the infected phenotype, including ones not detected by conventional cytopathicity and antibody-based assays. After deprioritization of confounding phospholipidosis, we present 74 hit candidates, including unreported compounds targeting host processes implicated in viral infection. This adaptable and scalable platform is suited for diverse viruses and cell systems. We provide a resource of open-access screening data, images, and analysis pipelines to advance antiviral discovery and pandemic preparedness.